Christina M. Martin

The prevalence and significance of occult hepatitis B virus in a prospective cohort of HIV-infected patients.

Background:Occult hepatitis B virus (HBV) is defined as low-level HBV DNA without hepatitis B surface antigen (HBsAg). Prevalence estimates vary widely. We determined the prevalence of occult HBV at the University of Cincinnati Infectious Diseases Center (IDC). Methods:Patients in the IDC HIV database (n = 3867) were randomly selected using a 25% sampling fraction. Samples were pooled for HBV nucleic acid extraction. Pools were tested for HBV DNA by a real-time polymerase chain reaction (PCR) assay to coamplify core/surface protein regions. The PCR assay was run on all individual samples from each DNA+ pool. DNA+ samples were tested for HBV serologic markers. Results:A total of 909 patients without known HBV were selected. The mean CD4 count was 384 cells/mm3. Forty-three patients were HBV DNA+. Twelve of 43 were DNA+/HBsAg− (95% confidence interval for database: 0.58% to 1.90%). Five of 12 were negative for all serologic markers. Alanine aminotransferase, aspartate aminotransferase, and HBV DNA titers were elevated in HBsAg+ patients versus occult patients and versus HIV-monoinfected patients. No other significant differences were detected. No occult HBV patient was on treatment with anti-HBV activity. Conclusions:Forty-three percent of those with HBV were not previously identified as HBV+, indicating the need for ongoing screening in high-risk populations. Occult HBV may occur in persons with all negative serologic markers, representing a challenge for identification.

Mutations Associated with Occult Hepatitis B Virus Infection Result in Decreased Surface Antigen Expression In Vitro

Summary.  Occult hepatitis B virus (HBV) infection is characterized by the absence of detectable hepatitis B surface antigen (HBsAg) in the serum, despite detectable HBV DNA. Investigations of the mechanisms underlying the development of occult HBV infection are lacking in the current literature, although viral mutations in the surface region, resulting in decreased HBsAg expression or secretion, represent one potential mechanism. Wild‐type HBsAg expression vectors were constructed from genotype‐matched chronic HBV sequences. Site‐directed mutagenesis was then utilized to introduce three genotype A mutations – M103I, K122R and G145A – associated with occult HBV infection in vivo, alone and in combination, into the wild‐type HBsAg vectors. Transfection of Huh7 and HepG2 cell lines was performed, and cell culture supernatants and cell lysates were collected over 7 days to assess the effects of these mutations on extracellular and intracellular HBsAg levels. The G145A mutation resulted in significantly decreased extracellular and intracellular HBsAg expression in vitro. The most pronounced reduction in HBsAg expression was observed when all three mutations were present. The mutations evaluated in vitro in the current study resulted in decreased HBsAg expression and potentially increased hepatic retention and/or decreased hepatic secretion of synthesized HBsAg, which could explain the lack of HBsAg detection that is characteristic of occult HBV infection in vivo.

Genomic variability associated with the presence of occult hepatitis B virus in HIV co-infected individuals.

Summary.  Occult hepatitis B virus (O‐HBV) infection is characterized by the presence of HBV DNA without detectable hepatitis B surface antigen (HBV DNA+/HBsAg−) in the serum. Although O‐HBV is more prevalent during HBV/HIV co‐infection, analysis of HBV mutations in co‐infected patients is limited. In this preliminary study, HBV PreSurface (PreS) and surface (S) regions were amplified from 33 HIV‐positive patient serum samples – 27 chronic HBV (C‐HBV) and six O‐HBV infections. HBV genotype was determined by phylogenetic analysis, while quasispecies diversity was quantified for the PreS, S and overlapping polymerase regions. C‐HBV infections harboured genotypes A, D and G, compared to A, E, G and one mixed A/G infection for O‐HBV. Interestingly, nonsynonymous–synonymous mutation values indicated positive immune selection in three regions for O‐HBV vs one for C‐HBV. Sequence analysis further identified new O‐HBV mutations, in addition to several previously reported mutations within the HBsAg antigenic determinant. Several of these O‐HBV mutations likely contribute to the lack of detectable HBsAg in O‐HBV infection by interfering with detection in serologic assays, altering antigen secretion and/or decreasing replicative fitness.

Cytokine expression during chronic versus occult hepatitis B virus infection in HIV co-infected individuals.

Chronic hepatitis B virus infection is characterized by persistent detectable levels of hepatitis B surface antigen (HBsAg) and HBV DNA in the serum. In contrast, HBsAg is not detectable during occult HBV infection, despite the presence of HBV DNA. An altered host immune response could play a role in the development of occult HBV infection; however, potential differences in immune responses among chronic and occult HBV-infected patients have not been evaluated in vivo. In the current study, we evaluated serum levels of regulatory, apoptotic, and fibrotic/anti-fibrotic cytokines/markers as indicators of immune responses in 25 chronic and 12 occult HBV-infected patients. More than half of the patients in both chronic and occult HBV infection groups had IL-2, IL-4, IL-13, and IFN-gamma levels below detectable limits. In contrast, most patients had detectable levels of IL-8, IL-10, IP-10, sFas, sFasL, and TGF-beta1. Of these, only sFas was significantly different between the two groups, with lower levels observed during occult compared to chronic HBV infection (p=0.01). As a surrogate marker of apoptotic inhibition, decreased sFas during occult HBV infection suggests that apoptosis occurs at different rates in occult compared to chronic HBV infection and therefore, may contribute to persistence of occult HBV infection.

Screening for hepatitis C virus non-nucleotide resistance mutations in treatment-naive women

OBJECTIVES Hepatitis C virus (HCV) non-nucleoside inhibitors (NNIs) target the viral RNA-dependent RNA polymerase encoded by the NS5B gene. Several NNIs share a similar allosteric binding site, and their antiviral efficacy is attenuated by a cysteine-to-tyrosine mutation at amino acid 316 (C316Y). In the current study, we assessed NS5B resistance mutations in treatment-naive individuals from a prospective natural history study of viral infections in women. METHODS Partial NS5B sequences from HCV-positive women were amplified by RT-PCR. Additionally, subcloning was performed to evaluate intrapatient variability in selected samples. RESULTS HCV NS5B genotypes were 45 genotype 1a (57.0%), 11 genotype 1b (13.9%), 5 genotype 2a (6.3%), 3 genotype 2b (3.8%), 9 genotype 3a (11.4%) and 6 genotype 4a (7.6%). One HCV genotype 1a-infected patient was found to have the C316Y mutation (1.3%). Clonal analysis further revealed that all NS5B sequences from this individual--representing three serum samples collected 4 years apart--contained the C316Y mutation. In contrast, the S282T resistance mutation was not found in any samples. CONCLUSIONS The C316Y polymerase resistance mutation was found in 1.3% of samples from HCV-infected women. The presence of this mutation over time suggests significant replicative fitness of this variant and has implications for development of new specifically targeted antiviral therapies against HCV (STAT-C) targeting this region.

Variability of the Polymerase Gene (NS5B) in Hepatitis C Virus-Infected Women

ABSTRACT There are limited data on diversity within the hepatitis C virus polymerase (NS5B). In concordance with its key functional role during the life cycle, NS5B intrapatient variability was low. Moreover, differences between NS5B nonsynonymous (dN) and synonymous (dS) mutation rates (dN − dS) were positively correlated with CD4 cell count, while nonsynonymous mutations were strongly correlated with reduced replication in vivo.

Analysis of a non-structural gene reveals evidence of possible hepatitis C virus (HCV) compartmentalization

Viral diversity is a hallmark of hepatitis C virus (HCV) infection; however, only limited data are available regarding HCV variability in extrahepatic sites, and none have systematically compared diversity in non‐structural and structural genomic regions. Therefore, HCV diversity in the NS5B and envelope 1 (E1) hypervariable region 1 (HVR1) genes was evaluated in matched sera and peripheral blood mononuclear cells (PBMCs) obtained from 13 HCV‐infected women. Multiple clonal sequences were compared to evaluate quasispecies diversity and viral compartmentalization in PBMCs. Genetic distances were higher for E1/HVR1 compared to NS5B in both the sera and PBMCs (P = 0.0511 and 0.0284). Genetic distances were higher in serum NS5B compared to PBMC NS5B (P = 0.0003); however, they were not different when comparing E1/HVR1 in sera to PBMCs. By phylogenetic analysis of NS5B, evidence of possible PBMC compartmentalization was observed for one woman, while statistical methods were consistent with PBMC compartmentalization for six women. Evidence of compartmentalization within a non‐structural genomic region may suggest that viral adaptation to a unique extracellular microenvironment(s) may be required for efficient replication and could contribute to HCV persistence. J. Med. Virol. 84:242–252, 2012.

Hepatitis B virus (HBV) X gene diversity and evidence of recombination in HBV/HIV co-infected persons.

The high frequency of mutation during hepatitis B virus (HBV) infection has resulted in 8 genotypes (A–H) with varying effects on disease severity and treatment efficacy. However, analysis of intrapatient HBV diversity is limited, especially during HIV co‐infection. Therefore, a preliminary study was performed to analyze HBV X gene diversity in 17 HBV/HIV co‐infected individuals. Phylogenetic analysis revealed HBV genotype A in 13 individuals (76.5%) or genotype E in 1 individual (5.9%). Additionally, 3 individuals were dually infected with HBV genotypes A and G (17.6%). Overall, higher genetic distance and entropy were observed in the X region and overlapping polymerase (Pol(X)) regions when compared to the PreS, S, and overlapping polymerase (Pol(PS) and Pol(S)) regions analyzed in the same patients as part of a previous study. In addition, multiple viral variants from 2 individuals with dual HBV infection did not group with either genotype A or G by phylogenetic analysis, indicating possible recombination. SimPlot bootscan analysis confirmed recombination breakpoints within the X gene in both individuals. Recombination between HBV genotypes may represent an important evolutionary strategy that enhances overall pathogenic potential and/or alters the downstream effects of the HBV X protein. J. Med. Virol. 83:1142–1150, 2011.

Evidence of distinct populations of hepatitis C virus in the liver and plasma of patients co-infected with HIV and HCV

Viral diversity is an important predictor of hepatitis C virus (HCV) treatment response and may influence viral pathogenesis. HIV influences HCV variability in the plasma; however, limited data on viral variability are available from distinct tissue/cell compartments in patients co‐infected with HIV and HCV. Thus, this exploratory study evaluated diversity of the hypervariable region 1 (HVR1) of HCV in the plasma and liver for 14 patients co‐infected with HIV and HCV. Median intra‐patient genetic distances and entropy values were similar in the plasma and liver compartments. Positive immune selection pressure was observed in the plasma for five individuals and in the liver for three individuals. Statistical evidence supporting viral compartmentalization was found in five individuals. Linear regression identified ALT (P = 0.0104) and AST (P = 0.0130) as predictors of viral compartmentalization. A total of 12 signature amino acids that distinguish liver from plasma E1/HVR1 were identified. One signature amino acid was shared by at least two individuals. These findings suggest that HCV compartmentalization is relatively common among patients co‐infected with HIV and HCV. These data also imply that evaluating viral diversity, including drug resistance patterns, in the serum/plasma only may not adequately represent viruses replicating with in the liver and, thus, deserves careful consideration in future studies. J. Med. Virol. 86:1332–1341, 2014.

Limited infection with occult hepatitis B virus in drug users in the USA.

Aim:  Occult HBV infection (O‐HBV) is defined as low level HBV replication in the absence of detectable circulating HBV surface antigen. O‐HBV has been implicated in HBV reactivation, advanced liver fibrosis and cirrhosis, reduced interferon response rates, elevated liver enzyme levels, and the development of hepatocellular carcinoma. However, the prevalence of O‐HBV has not been clearly established in certain at‐risk populations, such as injection drug users.